Osteopontin (Oldberg et al. (1986) Proc. Natl. Acad. Sci. USA 83:8819; Oldberg et al. (1986) J. Biol. Chem. 263:19433-19436) also known as OPN (Wrana et al. (1989) Nucl. Acid Res. 17:3306), 2ar (Smith and Denhardt (1987). J. Cell Biochem. 34:10-22), transformation-associated secreted phosphoprotein (Senger et al. (1989) Anticancer Res. 48:1291), or early T-lymphocyte activation-1 (Patarca et al. (1991) Proc. Natl. Acad. Sci. USA 88:2736), is a multifunctional secreted glycoprotein which is expressed by a wide variety of cell types including bone (Oldberg et al. (1986) J. Biol. Chem., supra), smooth muscle cells (e.g., cells of the vascular system) (Giachelli et al. (1991) Biochem. Biophys. Res. Commun. 177: 867-873), activated T-lymphocytes (Patarca et al. (1989) J. Exp. Med. 170:145-161; Patarca et al. (1991) Proc. Natl. Acad. Sci. USA, supra), macrophages (Singh et al. (1990). J. Exp. Med 171:1931-1942), and carcinomas and sarcomas (Senger et al., supra). In other tissues, osteopontin is expressed during various developmental stages and circulating levels of the protein have been found to be elevated in individuals with autoimmune diseases. Osteopontin is also elevated in sera from patients with advanced metastatic cancer and cellular transformation may lead to enhanced osteopontin expression and increased metastatic activity.
The protein is involved in a range of cellular functions including cell adhesion and spreading, cell migration and homing, chemotaxis, and calcium homeostasis (e.g., calcification). Osteopontin is induced by oxidative stress, including ischemia/reperfusion, heat shock or starvation, and exerts antioxidant effects by down-regulation of inducible nitric oxide synthetase (conferring protection against killing by macrophages). Moreover, osteopontin has been found to inhibit apoptosis in various cell types and in response to a wide range of stimuli.
In mammals, osteopontin is known to play an important role in regulation of bone formation and/or bone remodeling, regulation of immune responses, mediation of inflammation (e.g., tissue inflammation) in specific disease and injury states, angiogenesis, and arterial wound healing. Osteopontin has also been shown to be secreted by malignant tumors and is believed to play an important role in metastasis formation. The protein is subject to a large number of post-translational modifications (e.g., phosphorylation) and, in fact, the considerable number of functions that have been attributed to this protein are believed to be differentially regulated by such post translational modifications.
Osteopontin binds to cells via integrin and non-integrin receptors. The presence of a Gly-Arg-Gly-Asp-Ser (GRGDS, SEQ ID NO:1) cell-surface receptor binding motif within the sequence of osteopontin is involved in cell attachment and spreading via xcex1vxcex23, xcex1vxcex21 and xcex1vxcex25 integrins (Oldberg et al., supra). Cleavage of osteopontin with thrombin enhances its cell attachment properties. A distinct receptor-ligand interaction between CD44 and osteopontin, has also been shown to play a role in mediating chemotaxis and/or cell or attachment. Multiple phosphorylated and nonphosphorylated forms of osteopontin are secreted by cells and are differentially stimulated by tumor promoters (Kubota et al. (1989) Biochem. Biophys. Res. Commun. 162: 1453-1459). In addition, differential attachment of osteoclasts to surfaces coated with osteopontin isolated from various tissues and to phosphorylated and nonphosphorylated osteopontin has been demonstrated.
Given the important role that osteopontin plays in cellular processes including cell spreading and chemotaxis as well as the important functions it has in diverse processes including arterial wound healing, immune response, bone development, tissue remodeling, and metastasis, there exists a need to identify peptides and develop compounds that mimic or inhibit many of the unique functions of osteopontin. In particular, there exists a need for identifying peptides and developing compounds which mimic the chemotactic activities of osteopontin as well as agents (e.g., antibodies, peptides and compounds) which are inhibitory for osteopontin-dependent chemotaxis.
The present invention is based, at least in part, on the discovery of or identification of the chemotactic regions of the naturally-occurring osteopontin protein. This discovery led to the development of chemotactic compounds and peptides derived from osteopontin. The discovery also led to the development of peptides which are inhibitory to chemotaxis. Accordingly, the compounds and peptides of the present invention can be used to induce and/or inhibit chemotaxis, either in vivo or in vitro. The compounds and peptides of the present invention can be used to treat conditions or diseases associated with chemotaxis. For example, the compounds and peptides of the present invention can be used to treat or inhibit tumor metastasis, inflammation, osteoporosis and autoimmune disease. Moreover, the compounds and peptides of the present invention have applications in angiogenesis, wound-healing and in the development of prosthetic devices.
The present invention pertains to osteopontin derived compounds and peptides. The compounds and peptides are capable of modulating (e.g., inducing or inhibiting) the chemotaxis of several cell types. Examples of cell types include, but are not limited to, endothelial cells, periosteal cells, tumor cells, macrophages and osteoprogenitor cells.
In one embodiment, the invention features purified osteopontin-derived chemotactic peptide (e.g., purified osteopontin-derived peptides having chemotactic activity). In another embodiment, the invention features purified chemotactic compounds having the following formula:
Qxe2x80x94Axe2x80x94X,
wherein Q and X are flanking moieties and are absent or present and A is a hydrophobic core constituent, forming a compound having chemotactic activity.
In another embodiment, the invention features chemotactic compounds which include a hydrophobic core constituent (A) having the following motif:
"psgr"xe2x80x94xcex1xe2x88x92xe2x80x94xcex6xe2x80x94xcex2+,
wherein "psgr" is a hydrophobic patch, xcex1xe2x88x92 is an acidic moiety, xcex6 is a bend-forming moiety, and xcex2+ is a basic moiety.
The invention also pertains to isolated nucleic acid molecules encoding the osteopontin derived peptides of the present invention which can be used to produce the peptides and also as a therapeutic agent. Likewise, the invention pertains to antibodies (e.g., monoclonal antibodies) which specifically react with osteopontin-derived peptides. These antibodies can be administered to a subject in the form of a therapeutic composition to modulate the chemotactic effect of the peptides of the invention, thus neutralizing the migration of various cell types in response to osteopontin.
In another aspect, the invention features a therapeutic composition which includes an chemotactic compound, chemotactic peptide, inhibitory compound or peptide and a pharmaceutically-acceptable carrier or diluent. The therapeutic composition can be used in the methods described herein.
In another aspect, the invention features a method for modulating tumor invasion or tumor metastasis in a subject. In one embodiment, the method includes administering to a subject (e.g., at a tumor site) a therapeutically effective amount of an inhibitory compound, or a chemotactic peptide antibody, such that tumor invasion or tumor metastasis is modulated. In another embodiment, the method includes administering to a subject (e.g., at a tumor site) a therapeutically effective amount of an inhibitory compound complexed to a carrier (e.g., an extracellular matrix molecule, for example, collagen, glycosamoniglycans, for example, hyaluronic acid, chondroitin sulfates and heparan sulfates), such that tumor metastasis is modulated (e.g., inhibited).
In another aspect, the invention features a method for modulating nitrous oxide production in a cell or subject. In one embodiment, the method includes contacting a cell with an effective amount of a chemotactic peptide of the present invention (or an effective amount of an inhibitory peptide), such that nitrous oxide production is modulated (e.g., stimulated or inhibited, respectively). In another embodiment, the method includes administering to a subject a therapeutically effective amount of an chemotactic peptide or inhibitory compound such that nitrous oxide production is modulated (e.g., stimulated or inhibited, respectively).
In another aspect, the invention features a method for activating apoptosis in a cell or subject. In one embodiment, the method includes contacting a cell with an effective amount of a chemotactic peptide of the present invention such that apoptosis of the cell is activated. In another embodiment, the method includes administering to a subject a therapeutically effective amount of a chemotactic peptide such that apoptosis is activated (e.g., apoptosis of a cell(s) within the subject).
In another aspect, the invention features a method for promoting wound healing (e.g., scarless wound healing) in a subject. The method includes administering to a subject a therapeutically effective amount of a composition comprising an chemotactic compound or peptide and a pharmaceutically-acceptable carrier or diluent such that wound healing is promoted.
In another aspect, the invention features a method for promoting cell migration (e.g., cellular chemotaxis) to a target site. In one embodiment, the method includes administering to a subject (e.g. at a target site) a therapeutically effective amount of a chemotactic compound or peptide such that migration (e.g., cellular chemotaxis) of a desired cell to the target site is promoted. In another embodiment, the method includes administering at the target site, a chemotactic peptide or compound adhered to a substrate (e.g., an extracellular matrix components, for example, collagen, or glycosamoniglycans, including hyaluronic acid, chondroitin sulfates and heparan sulfates). In yet another embodiment, the method Includes coating a physical material (e.g., plastic, polyvinyl surface, steel, glass, polymer, PGA, metals, for example, titanium) with a chemotactic peptide prior to introducing the material to a subject, such that cell migration (e.g., cellular chemotaxis) is promoted.
In another aspect, the invention features a method for promoting cell migration (e.g., cellular chemotaxis) to a target site. In one embodiment, the method includes administering to a subject (e.g., at a target site) a therapeutically effective amount of a chemotactic compound or peptide such that migration (e.g., cellular chemotaxis) of a desired cell to the target site is promoted. In another embodiment, the method includes administering at the target site, a chemotactic peptide or compound adhered to a substrate (e.g., an extracellular matrix components, for ex.ample, collagen, or glycosamoniglycans. including hyaluronic acid, chondroitin sulfates and heparan sulfates). In yet another embodiment, the method includes coating a physical material (e.g., plastic, polyvinyl surface, steel, glass, polymer, PGA, metals, for example, titanium) with a chemotactic peptide prior to introducing the material to a subject, such that cell migration (e.g., cellular chemotaxis) is promoted.
In another aspect, the invention features a method for inhibiting cell migration (e.g., cellular chemotaxis) at a target site. In one embodiment, the method includes administering to a subject (e.g., at the target site) a therapeutically effective amount of an inhibitory compound or peptide or antibody such that migration (e.g., cellular chemotaxis) of a cell to the target site is inhibited. In another embodiment, the method includes administering at the target site, an inhibitory peptide or compound adhered to a substrate (e.g., an extracellular matrix components, for example, collagen, or glycosamoniglycans, including hyaluronic acid, chondroitin sulfates and heparan sulfates). In yet another embodiment, the method includes coating a physical material (e.g., plastic, polyvinyl surface, steel, glass, polymer, PGA, metals, for example, titanium) with an inhibitory peptide or compound prior to introducing the material to a subject, such that cell migration (e.g., cellular chemotaxis) is inhibited.
The invention also features a prosthetic device. The prosthetic device contains or coated with a therapeutically effective amount of a chemotactic or inhibitory compound or peptide in or on the prosthetic device.
The invention also features physical materials (e.g., plastic, polyvinyl surface, steel, glass, polymer, PGA, metals, for example, titanium) containing or coated with a therapeutically effective amount of a chemotactic or inhibitory compound or peptide.
In another aspect, the invention features a method for treating the formation of atherosclerotic plaques. The method includes administering to a subject a therapeutically effective amount of a chemotactic compound or peptide such that formation of artherosclerotic plaques is prevented.
In another aspect, the invention also features a method for treating an angiogenic-associated disease. The method includes administering to a subject a therapeutically effective amount of an antibody specifically reactive with a chemotactic peptide, or an inhibitory compound or peptide such that treatment of angiogenic-associated disease occurs.
In yet another aspect, the invention features a method of inducing in vitro chemotaxis of a cell. The method includes exposing the cell to a chemotactic compound or peptide in an amount effective to induce chemotaxis, such that chemotaxis is induced. In yet another aspect, the invention features a method of inhibiting in vitro chemotaxis of a cell. The method includes exposing the cell to an inhibitory compound or peptide in an amount effective to inhibit chemotaxis, such that chemotaxis is inhibited.
The methods of the invention are particularly useful for modulating the migration of cells (e.g., cells the movement of which it is desirable to control) involved in wound healing (e.g., extracellular matrix cells (connective tissue cells) involved in wound healing) to thereby promote recovery from wounds. The methods of the invention are particularly useful for modulating the migration of neoplastic cells (e.g., carcinoma cells, for example, of breast, testis, ovary, lung, gastrointestinal tract) to thereby modulate (e.g., inhibit) spreading from one location to another.
Antibodies specifically reactive with the chemotactic peptides of the invention or inhibitory compounds and/or peptides can also be administered to a subject having a metastatic disease (e.g., cancer) to modulate tumor invasion or to prevent or inhibit metastasis of the disease by inhibiting the chemotactic activity of osteopontin. The peptides, compounds and antibodies can be administered to the subject in the form of a therapeutic composition which includes the peptide, compound or antibody and a pharmaceutically acceptable carrier or diluent.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.